Complex oligosaccharides have been proposed to exert important biological properties for human health. However, despite advances in the commercialization of simple plant oligosaccharides and polysaccharides, these products of plant metabolism are unable to substantially alter the human gut microbial ecology nor its metabolism nor the physiology and disease risks of humans. Human milk, on the other hand, is an attractive source of oligosaccharides precisely because human milk produces a unique microbiota whose persistence yields scientifically established evidence of benefits to human infants. The free oligosaccharides in human milk have emerged through evolution in remarkable abundance and with significant structural diversity. Research in the collaborating laboratories has demonstrated that these oligosaccharides constitute a symbiotic system in human infants stimulating the competitive growth of commensal bifidobacteria. In this project we propose to build the analytic, genetic and biological tools to establish the structures and functions of the oligosaccharides in human milk as thematic principles to guide the development, research and industrialization of bioactive oligosaccharides for human health. To achieve this goal, we will: (A) elucidate in precise chemical detail the entire human milk glycome and categorize a diverse pool of human lactating subjects differing in the composition of expressed oligosaccharides, (B) develop analytical tools to rapidly determine human secretor versus non- secretor status based on the oligosaccharide profile and link maternal secretor status to milk oligosaccharide composition and microbiota diversity, and (C) establish the genetic basis of bifidobacterial selection by the specific structures in the ensemble of human milk oligosaccharides using whole genomic analysis of a range of bifidobacterial strains, relating specific oligosaccharide consumption by bacteria to functional analysis of genes and proteins in and on bacterial surfaces.